KR970011555B1 - Green tea extract im antithrombotic agent and a process for preparing the same - Google Patents

Abstract

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Description

Green tea extract contained as an active ingredient in an antithrombotic agent and its preparation method

1 to 4 are graphs showing the aggregation inhibitory effect when the green tea extract of the present invention is administered with ADP, epinephrine, collagen or ristocetin, the aggregation promoter.

Figure 5 is a graph showing the thrombus inhibitory effect when the administration of the green tea extract of the present invention, combined administration of epinephrine and collagen, the aggregation promoter first.

Figure 6 is a graph showing the bleeding time when the green tea extract of the present invention and the control aspirin.

7 is a graph comparing the whole blood coagulation time when the green tea extract of the present invention and the control drug aspirin were administered.

8 is a graph showing the plasma coagulation time when the green tea extract of the present invention and the control drug aspirin is administered.

[Purpose of invention]

[Technical Field to which the Invention belongs and Prior Art in the Field]

The present invention relates to a green tea extract contained in the anti-thrombotic agent as an active ingredient and a method for producing the same. More specifically, the present invention hot water extract the dried green tea leaf with water or a mixture of water and lower alcohol, and filtration and / or centrifugation without chloroform extraction process to remove the non-polar material and then column chromatography By lyophilization, the present invention relates to a green tea leaf extract powder comprising 25-30% of polyphenols, 40-70% of epigallocatechin calate and 0-35% of a mixture of epicatechin and epigallocatechin, and a preparation method thereof.

Green tea is one of the favorite beverages in the Far East, mainly in China and Japan.

The components present in the green tea leaves include green tea tannins, caffeine, ascorbic acid, tocopherols, hulabonols, polysaccharides and beta carotene, among which caffeine, ascorbic acid and green tea tannins. Caffeine and ascorbic acid are also included in other plant-derived teas, but green tea tannins, represented by catechins, are unique ingredients found only in green tea, such as epicatechin (EC) and epicatechin gallate (epicatechin gallate). ECG), epigallocatechin (EGC), and epigallocatechin gallate (EGCG) contain four kinds as main components, and other fluorine, zinc, calcium and magnesium salts.

The main effects of green tea catechins include the inhibition of lipid peroxidation, the inhibition of free radicals in various diseases, the mutagenicity of carcinogens, the inhibition of cholesterol resorption, the antibacterial and antiviral effects, the prevention of tooth decay and deodorization. Several effects are known. Especially in the 1990s, various results have been reported, such as the induction process of carcinogens and the inhibitory action on carcinogens by EGCG among these components, and the development of anticancer drugs from green tea catechins has been reported. Being potent.

In addition, several researchers have extracted pharmacological components with platelet aggregation inhibitory ability from green tea. For example, Wang et al. Have reported that polysaccharides extracted from green tea have the effect of thrombolysis on laboratory and in vivo conditions (see Wagn Shuru et al., Zhoogcaoyao, 23 (5): 254-256). (1992), Shen et al. Reported that polyphenols extracted from green tea reduced the formation of blood clots under laboratory conditions (Shen Xinnan et al., Yingyang Xuebao, 15 (2): 147-151). (1993)).

In addition, Japanese Patent Application Laid-open No. Hei 2-184626 adds 20-40 L / kg of hot water to green tea leaves, oolong tea leaves, or black tea leaves, leaches it for 10-30 minutes, concentrates and freeze-dried to obtain a soluble extract, The leaves were hydrothermally extracted, followed by chloroform extraction to remove natural polymers such as caffeine, and then extracted again with ethyl acetate to obtain crude catechins. Thus obtained soluble extract or crude catechins were subjected to column chromatography using acetone, a hydrophilic organic solvent, as the elution solvent, thereby obtaining the most preferred tea-catechin fraction containing 30% ECG and 70% EGCG. It has been demonstrated experimentally that ECG and / or EGCG obtained as described above, soluble extract or tea-catechins comprising this component have higher platelet aggregation inhibitory ability than commercial antithrombotic aspirin, and as their platelet aggregation inhibitor The use is disclosed.

However, the amount of ECG and EGCG included in the soluble extract obtained by the method disclosed in the above document was about 30%, indicating a low content. In addition, the document did not investigate the effect of inhibiting platelet aggregation on various platelet aggregation promoters, and mainly investigated collagen inhibitory effect only on collagen, and did not investigate the anti-thrombotic effect and anti-blood coagulation ability in vivo. Therefore, the green tea extract obtained by the above-described method has a disadvantage that it is not suitable as a platelet aggregation inhibitor.

On the other hand, the catechins separation process of the green tea generally used in the prior art can be divided into three steps: 1) extraction process, 2) removal process and 3) purification process. Conventional extraction process uses a mixture of hot water, cold water, methanol or ethanol and a suitable combination thereof, and the process of removing other components other than the active ingredient from the extract is mainly an organic solvent that does not mix with water extraction and removal effect In order to maximize the maximal polarity, chloroform, ethyl acetate, diethyl ether and the like having large polarity are used. In the final purification process, a partition extraction method using different polarities of organic solvents is mainly used. Recently, column chromatography or high performance liquid chromatography has been used. The purification process by the partition extraction method takes a long time in the extraction operation, because there is a large amount of organic solvents such as chloroform, methyl isobutyl ketone, there is a problem in economy and stability during the separation operation. Sepadex or polymer-based resins are used for the purification method by column chromatography, but column resins, elution solvents, elution conditions, etc., which can provide optimal green tea catechins separation in connection with the aforementioned extraction and removal processes Is different and its optimization process requires considerable experimentation. Purification by high-performance liquid chromatography has the advantage of being able to purify the product quickly and efficiently, but has problems such as the purchase of a measuring device, the high cost of an eluting solvent, and deterioration of a separation column.

[Technical problem to be achieved]

Accordingly, the present inventors have made intensive research to solve the above-mentioned problems. As a result, the hot water extract was first completely removed from the residue by filtration or centrifugation, Sephatex LH-20 column chromatography, and the separation solvent were simplified to obtain water and By washing once with acetone, dioxane, methanol or isopropane in a mixed solution, it was possible to completely wash not only water-soluble caffeine, free amino acids and sugars, but also tepenoids and coloring components. Therefore, there is no need for a separate decaffeine operation as in the conventional method, and by using organic solvents such as chloroform and methyl isobutyl getone, it is possible to prevent incorporation of organic solvents or contaminants derived from organic solvents appearing in the final product. have. As a result, only the red-yellow portion is present at the tip of the column, which can be easily observed with the naked eye. On the other hand, the remaining red-yellow portion can be eluted at once by water-acetone or dioxane mixed solvent to obtain the active ingredient in the maximum yield, it was able to completely escape from the drawbacks of the known method by the method of the present invention.

In addition, the dried green tea leaves were extracted with hot water with water or a mixture of water and lower alcohol, centrifuged without chloroform extraction process to remove nonpolar substances, followed by ethyl acetate extraction, and then a mixture of water and acetone, dioxane, methanol or isopropanol. Green tea extract prepared by column chromatography using a separate solvent and freeze-dried the active fraction contained 25-30% polyphenols, 40-70% EGCG, 0-35% mixture of EC and ECG, the extract The ADP, epinephrine, collagen, ristocetin (platelet aggregation) to a variety of platelet aggregation promoting substances, such as the effect of inhibiting platelet aggregation is excellent, and also in vivo anti-thrombotic effect and blood coagulation performance was confirmed that the present invention, Completed.

After all, the main object of the invention is the above-mentioned hot water extraction, filtration and / or centrifugation, ethyl acetate extraction, column chromatography and lyophilization, polyphenols 25-30%, EGCG 40-70%, a mixture of EC and ECG 0 It is to provide a method for producing green tea leaf extract powder containing -35%.

Another object of the present invention is to provide an anti-thrombotic agent containing the green tea leaf extract powder prepared by the electric method as an active ingredient and an adjuvant such as excipients, diluents and antioxidants commonly used in the manufacture of pharmaceutical preparations. .

[Configuration and Function of Invention]

Hereinafter, the present invention will be described in more detail.

In the present invention, the dried green tea leaves are extracted with hot water with water or a mixture of water and lower alcohol, filtered and / or centrifuged without chloroform extraction to remove non-polar substances, and concentrated, followed by repeated extraction with ethyl acetate. Then, using a mixed solution of water and acetone, dioxane, methanol or isopropanol as a separation solvent, column chromatography, and the obtained active ingredient is lyophilized to prepare a green tea leaf extract powder (green tea extract). In the above, when the extract is concentrated, n-butanol must be added to about 1/4 of the extract in consideration of the effective antioxidant effect, so that the extract is always exposed to the air at the top of the extract so that it is not oxidized. Maintain volume.

As a result of preparing the green tea extract by the above-described method, it was possible to remove water completely, and the composition ratio of the concentrated liquid and the composition ratio of the active ingredient after lyophilization were almost unchanged. The dried active ingredient powder was grayish brown, soluble in water, and very stable because it was not hygroscopic even when stored for 6 months or longer. Thus dried powders can be easily prepared into pharmaceutical preparations.

As a result of repeating the above method, the composition of each component of the product containing the active ingredient according to each batch was almost identical. As a result of examining the composition of the active fraction, it contained 25-30% polyphenols, 40-70% epigallocatechin gallate, and 0-35% mixture of epicatechin and epigallocatechin. This is very different from the composition of the green tea extract having platelet aggregation inhibitory ability, which has been separated by the extraction method reported so far.

The general extraction method of the green tea extract of the present invention is described in more detail by the following methods.

Extraction Method

The dried green tea leaves as a starting material are extracted as is or Deok-eum tea with hot water of 70-90 ° C. for at least 10 minutes. At this time, the hot water used for extraction is used in an amount of 5-100 times, preferably 10-30 times per weight of green tea leaves, the extraction time depends on the extraction temperature, but generally 30 seconds to 1 hour, preferably 5-20 minutes. The composition of the hydrothermal extraction solvent may be used by mixing an alcoholic solvent of about 5-50%.

The extraction mixture is preferably completed in a range that does not boil while stirring. Insoluble matter in the extract is filtered by known methods such as subscription filtration, suction filtration or centrifugation to obtain a filtrate.

Condensation method under reduced pressure

About 10-50% (v / v), preferably about 20%, of n-butanol is added to the extract obtained by the extraction method, and the ratio is maintained until the concentration is completed under reduced pressure, thereby preventing boiling of the extract. On the other hand, by preventing the direct contact of the active ingredients in the air with the extract, to prevent changes in the composition of the composition.

Concentration is carried out under reduced pressure, for example at 10-70 mmHg, while heating at about 50-70 ° C. to produce a viscous extract (water content of about 10-30% (w / w)). The time required for concentration under reduced pressure depends on the degree of reduced pressure, but is about 30 minutes-2 hours. In the concentration method under reduced pressure, the concentration temperature does not rise above 90 ° C. to prevent loss or destruction of the active ingredient in the extract.

Ethyl acetate extraction, column chromatography and freeze drying method

The vacuum concentrate was repeatedly extracted several times with ethyl acetate, the organic solvent layer was concentrated, and column chromatography, most preferably Sephadex LH-20 column chromatography. At this time, the separation solvent is a mixed solution of water and acetone, dioxane, methanol or isopropanol.

The purified liquid obtained from the column is concentrated under reduced pressure at room temperature without applying heat, and then rapidly frozen and stored. In the freeze-drying method, the sample is left in a vacuum state at a cryogenic state by a refrigerant of -100 ° C. or lower, thereby completely removing sublimable material and remaining water. The drying time depends on the sample volume, surface area, refrigerant temperature and the capacity of the vacuum pump, but it is possible to obtain a completely dry product within about 12-24 hours.

Since the dry powder thus obtained is a hygroscopic and low-cost powder, it is suitable for preparing a pharmaceutical preparation such as powder, microgranules, granules or tablets according to known methods.

In addition, the dry powder thus obtained is less toxic and can be administered orally. The dried powder may contain pharmaceutically acceptable excipients (e.g., starch, lactose, calcium carbonate, calcium phosphate, etc.), binders (e.g., starch, gum arabic, carboxymethylcellulose, hydroxymethylcellulose, crystalline cellulose, etc.) Capsules, powders, granule microgranules or tablets, soft by mixing with lubricants (e.g. magnesium stearate, talc, etc.) and disintegrating agents (carboxymethylcellulose calcium, talc synthetic aluminum silicates, etc.), diluents (water, vegetable oils, etc.) Pharmaceutical preparations such as capsules and liquids.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1

Preparation of Green Tea Extract (I)

Green tea leaf dried, Deokeum tea or 200g of commercial green tea was extracted with hot water 4.000ml at 70-90 ° C for 20 minutes. At this time, Deokeum tea refers to the semi-finished product before the production of green tea by cutting the dried green tea leaves. The obtained extract was first filtered to filter out insoluble matters, and the filtrate was further centrifuged at 100 rpm for 10 minutes to remove precipitated residue. The upper filtrate (about 3,500 ml) was collected, concentrated under reduced pressure at 70 ° C. to about 400 ml using a vacuum rotary evaporator, and extracted with 400 ml of chloroform to completely remove nonpolar compounds. Such extraction was carried out three or more times. Subsequently, about 1,200 ml of the organic solvent layer obtained by repeatedly extracting the aqueous layer three times with 400 ml of ethyl acetate was concentrated to 50 ml at 50 ° C. using a vacuum rotary evaporator. Then, 200 ml of acetone or dioxane was added to the concentrate, and the resultant was concentrated to 20 ml. 20 ml of the concentrate was injected into the top of a Sephadex LH-20 column (35 mm in diameter x 550 mm in length, 200 g of Sephadex weight), followed by 3,000 ml of a solution of water-acetone or water-dioxane (84: 15, volume ratio). After washing, if it is determined that the greenish yellow and brown layers in the column are completely eluted, the yellowish brown to yellow bands adsorbed on the column upper layer start to elute with water-acetone or water-dioxane mixed solution (40:60, volume ratio). When the band) began to elute at the top of the column, the fraction was collected and concentrated to 70 ml at 70 ° C. using a vacuum evaporator. The concentrate was stored at −20 ° C. or less for 1 day, and then dried in a small amount in a lyophilizer to obtain 8.6 g of dry powder (EGCG residual ratio: 70%).

5.0 mg of the dried powder obtained by the above method was precisely taken up and dissolved in 5.0 mg of methanol, and 5 µl of this was subjected to high performance liquid chromatography, and the content of EGCG was measured. As a result, the residual ratio of EGCG was 70%. At this time, the residual ratio of EGCG was calculated by the following equation.

Residual ratio (%) of EGCG = corresponding peak area / total peak area × 100

<High-speed liquid chromatography measurement conditions>

Measuring instrument: High speed liquid chromatography (P-6300, Hitachi, Japan)

Elution conditions: Acetonitrile-ethyl acetate-0.05% phosphoric acid (12: 2 86, v / v) mixed solvent

Separation column: Cosmosil 5C18 (4.6mm i.d. × 150mm L) (Nakalai Tesque, Japan)

Column Temperature: 40 ℃

Wavelength: 280nm

Under these conditions, the residence time of EGCG was 6.9 minutes.

The residual ratio of EGCG of the dry powder obtained in Examples 2 to 6 to be described later was also evaluated in the same manner as described above.

Example 2

Preparation of Green Tea Extract (II)

1.5 kg of dried green tea leaves, Deok-eum tea or commercial green tea were extracted with 20.0 liters of a water-ethanol (90:10, volume ratio) mixed solution at 70 ° C. for 20 minutes, and the extract was first filtered using a filter paper to remove insoluble matters. The filtrate (about 19.0 liters) was collected and concentrated under reduced pressure at 70 ° C. using a vacuum rotary evaporator to obtain a viscous extract. The viscous extract was dissolved again in 2.0 liters of hot water, and then divided into 500 ml portions of a chloroform-n-hexane mixed solvent (80: 20, Volume ratio) to 500 ml to completely remove the non-polar compounds. The extraction removal operation was performed three or more times. Subsequently, approximately 1,500 ml of the organic layer obtained by repeating extraction of the aqueous layer three times with 500 ml of ethyl acetate was completely dried at 50 ° C. using a vacuum rotary evaporator. 500 ml of acetone was added to the dried product, and the resultant was concentrated to 50 ml. 50 ml of the concentrate was injected into the top of a Sephadex LH-20 column (70 mm in diameter x 500 mm in length, 400 g of Sephadex net weight), the water-soluble substance was removed with 4.0 liters of water, and then a solution of water-acetone (70: 30, volume ratio) 3.0 If it is determined that the green yellow and brown layers in the column have been completely eluted by washing with a liter, the greenish yellow and brown layers are eluted with a water-acetone (40:60, volume ratio) solution. When it started to receive the fractions and concentrated to 70ml at 70 ℃ by vacuum evaporation dryer. The concentrated solution was stored at -20 ° C. or less for one day and then completely dried in a lyophilizer to obtain 43.8 g of dry powder (EGCG residual ratio 54%).

Example 3

Preparation of Green Tea Extract (III)

Green tea leaf dried, Deokeum tea or commercial green tea 200g extracted with hot water of 70-90 ℃, 4,000ml for 20 minutes, the extract was filtered first to filter out the insoluble matter, and the filtrate was precipitated again by centrifugation at 2000rpm for 10 minutes. The residue was removed. The upper filtrate (about 3,500 ml) was collected and concentrated under reduced pressure until it reached about 400 ml at 70 ° C. with a vacuum rotary evaporator. Then, about 1,200 ml of the organic solvent layer obtained by extracting three times with 400 ml of ethyl acetate were subjected to a vacuum rotary evaporator. Concentrated to 50 ml at ℃. 200 ml of isopropanol was added to the concentrate, concentrated to 20 ml, and then 20 ml of the concentrate was poured on top of a Sephadex LH-20 column (35 mm diameter x 550 mm in length and 200 g of Sephadex net weight), followed by water-acetone (85:15, 3,000 ml of volumetric solution, and when it is judged that the greenish yellow and brown layers in the column are completely eluted, it starts to elute with 5.0 liters of water-acetone (55:45, volume ratio) solution, and the yellowish brown adsorbed on the column upper layer. When the yellow band began to elute at the front of the column, the fraction was received and concentrated to 70 ml at 70 ° C. with a vacuum evaporator. The Sephadex LH-20 column can be reused after eluting the non-polar material remaining in the column with 2.0 liters of acetone. The concentrate was stored at −20 ° C. or less for 1 day, and then dried in a small amount in a lyophilizer to obtain 14.7 g of dry powder (EGCG residual ratio: 62%).

Example 4

Preparation of Green Tea Extract (IV)

100 g of dried green tea leaves, Deok-eum tea or commercial green tea were extracted 1.0 liter of hot water at 70-90 ° C. for 10 minutes, and the extract was first filtered to filter out the insoluble matter, and the filtrate was again centrifuged at 2000 rpm for 10 minutes to settle the residue. Was removed. The upper filtrate (approximately 0.8 liters) was collected and concentrated under reduced pressure until it became about 100 ml at 70 ° C. with a vacuum rotary evaporator. Then, about 280 ml of the organic solvent layer obtained by extracting three times with 100 ml of ethyl acetate was repeatedly heated at 50 ° C. with a vacuum rotary evaporator. Concentrated to viscous state. 150 ml of methanol was added to the concentrate, concentrated again to 15 ml, and then 15 ml of the concentrate was poured onto the top of a Sephadex LH-20 column (35 mm in diameter x 550 mm in length, 200 g of Sephadex net weight), and water-methanol (85:15, When the volume of the solution was sufficiently washed with 4.0 liter of the volume ratio, and the green yellow and brown layers in the column were completely eluted, the solution began to elute with water-methanol (30:70, volume ratio) solution, and the yellowish brown to yellow color adsorbed on the upper layer of the column. When the band began to elute at the front of the column, the fraction was received and concentrated to 70 ml at 70 ° C. with a vacuum evaporator. The concentrated solution was stored at -20 ℃ or lower for 1 day and then completely dried in a lyophilizer to obtain 5.5g of dry powder (EGCG residual ratio: 58%).

Example 5

Preparation of Green Tea Extract (V)

Green tea leaf dried, Deokeum tea or commercial green tea 200g extracted with hot water 4,000ml at 70-90 ℃ for 20 minutes, the extract was filtered first to filter out the insoluble matter, and the filtrate was again centrifuged at 2000rpm for 10 minutes to precipitate Was removed. The upper filtrate (about 3,500 ml) was collected and concentrated under reduced pressure until it reached about 400 ml at 70 ° C. with a vacuum rotary evaporator. Then, about 1,200 ml of the organic solvent layer obtained by extracting three times with 400 ml of ethyl acetate were subjected to a vacuum rotary evaporator. Concentrated to 50 ml at ℃. 200 ml of isopropanol was added to the concentrate, and the resultant was concentrated to 20 ml. Then, 20 ml of the concentrate was poured on top of a Sephadex LH-20 column (35 mm in diameter x 550 mm in length and 200 g of Sephadex net weight), followed by water-isopropanol (90:10, 10, 2.5 liters of solution of volume ratio, and when it is judged that the greenish yellow and brown layer in the column was completely eluted, the yellowish brown to yellow band which began to elute with water-isopropanol (50:50, volume ratio) solution and was adsorbed on the column upper layer. When (band) began to elute at the top of the column, the fractions were received and concentrated to 70 ml at 70 ° C. with a vacuum evaporator. The concentrated solution was stored at -20 ° C or lower for 1 day, and then completely dried in a small amount in a freeze dryer to obtain 10.2 g of dry powder (EGCG residual ratio: 70%).

Example 6

Preparation of Green Tea Extract (VI)

200 g of dried green tea leaves, Deok-eum tea or commercial green tea were extracted with 40 liters of 40-65 ° C. water-ethanol (50:50, volume ratio) solution for 60 minutes, and the extract was filtered first to filter out the insoluble matters, and then the filtrate was filtered. Again, the precipitated residue was removed by centrifugation at 2000 rpm for 10 minutes. The upper filtrate (about 3.0 liters) was collected and concentrated under reduced pressure at 70 ° C. with a vacuum rotary evaporator. Then, the concentrated dried material was dissolved in 500 ml of a water-methanol (80:20, volume ratio) mixed solution, and then dissolved in 400 ml of ethyl acetate. About 1,200 ml of the organic solvent layer obtained by the repeated extraction three times was concentrated by vacuum rotary evaporator to 50 ml at 50 ° C. 200 ml of dioxane was added to the concentrate, concentrated again to 20 ml, and then 20 ml of the concentrate was poured on top of a Sephadex LH-20 column (35 mm in diameter x 550 mm in length, 200 g of Sephadex net weight), and water-dioxane (85: 15, volume ratio), when it is determined that the green yellow and brown layers in the column have been completely eluted by rinsing with 3,000 ml of the solution, the yellowish brown adsorbed on the column upper layer begins to elute with water-dioxane (40:60, volume ratio) solution. When the yellow band began to elute at the front of the column, the fraction was received and concentrated to 70 ml at 70 ° C. with a vacuum evaporator. The concentrated solution was stored at -20 ° C or lower for 1 day, and then completely dried in a small amount of lyophilizer to obtain 7.8 g of dry powder (EGCG residual ratio: 55%).

The platelet aggregation inhibitory ability of the dry powder obtained in Examples 1 to 6 was measured. Blood coagulation is divided into primary hemostasis in which platelets, which are active ingredients in the blood, and secondary hemostasis in which so-called plasma components, except active ingredients, are involved. Among them, primary hemostasis is classified to prevent thrombus formation and prevent blood clotting. In order to prevent the formation of blood clots and prevent blood clotting, the development of primary hemostatic blockers can be said to be of value as a fundamental therapeutic agent.

Experimental Example

Effect on Primary Hemostasis

1. Platelet aggregation inhibition test

The antiplatelet aggregation effect was investigated by the Turbidimetric method by Bonn using Aggrecorder II Pa-3220 (Kyoto Daiichi Kagaku Co. Ltd.). The principle of investigation is to adjust the platelet number of platelets rich plasma (PRP) to about 200,000-400,000 / ㎣, and the aggregation promoters (ADP, epinephrine, collagen, ristocetin) And agglomeration to observe the change in absorbance (OD) resulting from the aggregation.

Investigations were performed using a 1: 9 ratio in a 3.8% sodium citrate tube with anticoagulant from healthy persons with normal platelet function. To obtain PRP, centrifugation was performed at 160 × g (1,000 rpm) for 10 minutes and the platelet count was adjusted to 200,000-400,000 / dl. In order to obtain pallet poor plasma (PPP), centrifugation was further performed at 2,000 × g (3,400 rpm) for 10 minutes. The test was carried out using Aggrecorder II Pa-3220, and the temperature was fixed at 37 ° C. and the rotation speed was 1,000 rpm, and the platelet-deficient plasma was corrected. The platelet-rich plasma and the sample were transferred to a measuring instrument, left for 3-5 minutes in advance, and platelet aggregation promoting substance was added, and then the aggregation process was measured for 10 minutes.

As a result of the platelet aggregation test described above, the green tea extract showed an effect of strongly inhibiting platelet aggregation by all aggregation promoters at 1 mg / ml. The inhibitory effect was 60% in ADP, 96.5% in epinephrine, 35.3% in collagen, and 36.5% in ristocetin. On the other hand, the commercial antithrombotic Aspirin (0.1mM) showed a 13.5% inhibitory effect on ADP, 79.5% inhibition on epinephrine, and 72.6% inhibition on collagen. There was no inhibitory effect at all.

1 is a graph showing the coagulation inhibitory effect when administering the coagulation promoter ADP and the green tea extract of the present invention, Figure 2 is a graph showing the coagulation inhibitory effect when administering the coagulation promoter epinephrine and the green tea extract of the present invention 3 is a graph showing the coagulation inhibitory effect when the collagen and the green tea extract of the present invention is administered, and FIG. 4 is the coagulation inhibitor when the green tea extract of the present invention and ristocetin, the coagulation promoter, are administered. It is a graph showing the effect. From the results of this experiment, the green tea extract of the present invention was found to have an excellent platelet aggregation inhibitory effect.

2. Determination of antithrombotic action in vivo

Induction of experimental thrombi was performed according to the experimental method such as Dimino. At this time, the mouse is a male ICR mouse weighing about 20-30g, the induction of thrombus is injected into the tail vein of the platelet aggregation promoters (epinephrine, collagen) to form rapidly in the microvessels, used aggregation The promoter was prepared to contain 15 μg of collagen and 400 μM of epinephrine in 100 μl of saline, and injected into the tail vein at a dose of 100 μl per 20 g of mouse weight.

In order to examine the antithrombotic effect of the green tea extract, 100 mg and 10 mg of the sample were administered per 1 kg of mouse weight 2 hours before the experiment, and the total dose was 0.4 ml or less. The antithrombotic effect of green tea extract is calculated as a percentage of the number of experimental animals protected from paralysis or death of mouse hind limbs caused by the administration of platelet aggregation promoters, where paralysis is lost or trembling for more than 15 minutes. When was continued.

As a result of determining the concentration of epinephrine and collagen that induces an appropriate thrombus, 95% of blood clots could be induced by intravenous injection of 15 μg of collagen and 40 μM of epinephrine in 100 μl of saline. Thrombosis was determined by the presence of thrombocytopenia and hind limb paralysis lasting more than 15 minutes after administration of platelet aggregation promoting agent. The criterion was based on the state of tremor phenomena accompanied by hind limb paralysis.

As a result of the anti-thrombosis test, 100 mg of green tea extract per kg body weight of the mouse was intraperitoneally administered, showing 66% of the preventive effect, and 10mg of the intraperitoneal administration showed 44% of the protective effect. On the other hand, aspirin (100mg / Kg), a control drug, showed a 45% protective effect.

Figure 5 is a graph showing the antithrombotic effect when the first administration of epinephrine and collagen, the aggregation promoter, and the green tea extract of the present invention.

From the results of this experiment, it was confirmed that the green tea extract of the present invention has an excellent antithrombotic effect, which is equivalent to aspirin at a dose of about 1/10 of aspirin.

3. Blood Coagulation Test

(1) Measurement of Bleeding Time

Samples were administered to 180-200 g male Sprague-Dawley rats once a day for 10 days. Anesthetized by intraperitoneal administration of sodium pentobarbital (400mg / Kg) in rats, cut 5mm at the tip of the tail, and soak in saline solution at 37.5 ℃ to 5cm of the tail for hemostasis. The time until was measured.

The results showed that the bleeding time was 65.0 ± 12.9 seconds in the control group without drug administration, 165.1 ± 34.3 in the control aspirin (100 mg / kg), 113.2 ± 19.6 seconds in the 10 mg / kg green tea extract group, and 250.7 at 100 mg / kg. Significantly and dose-dependently increased to 509.8 ± 1.6 seconds at ± 117.3 seconds at 300 mg / kg. Therefore, the green tea extract of the present invention was found to show a longer bleeding than aspirin (see Fig. 6). 6 is a graph showing the bleeding time when the green tea extract of the present invention and the control drug aspirin is administered.

(2) Measurement of Whole Blood Clotting Time

One day after the bleeding time was measured, rats were anesthetized with ether, and 5 ml of blood was taken from the heart with a plastic syringe pre-filled with 0.5 ml of 3.13% sodium citrate solution. The collected blood was gently mixed, collected in a syringe, 1 ml of blood was placed in a glass test tube, and 200 μl of 1.7% CaCl ₂ · 2H ₂ O was added thereto, and the mixture was shaken at room temperature to measure the time until blood clots formed.

Experimental results showed that the whole blood clotting time in the control group without drug administration was 109.4 ± 14.0 seconds, and 137.2 ± 19.4 in the control aspirin (100mg / kg), whereas in the green tea extract group, 130.1 ± 20.5 seconds, 100mg / The dose-dependent increase was 140.9 ± 14.2 seconds in kg and 150.6 ± 17.6 seconds in 300 mg / kg (see Fig. 7). 7 is a graph comparing the whole blood clotting time when the green tea extract of the present invention and aspirin were administered.

(3) Measurement of plasma clotting time

Part of the blood obtained above was centrifuged at 25,000 rpm to obtain plasma, and 100 μl of plasma, 100 μl of saline, and 50 μl of 50 mM CaCl ₂ were placed in a glass test tube, and the time until a clot was formed was stirred at 37 ° C. .

The experimental results showed that the plasma coagulation time in the control group without drug administration was 146.2 ± 26.5 seconds, 222.5 ± 40.2 in the control aspirin (100mg / kg), 177.5 ± 37.6 seconds in the 10mg / kg green tea extract group, and 100mg / kg. Dose-dependently increased from 219.8 ± 57.9 sec and 300 mg / kg to 233.1 ± 83.2 sec (see FIG. 8). 8 is a graph showing the plasma coagulation time when the green tea extract and aspirin of the present invention are administered.

4. Inhibition of supporting peroxidation and efficacy in platelets

a) Determination of the inhibitory effect of malondialdehyde (MDA) production

1 ml of platelet suspension was added to the isotonic solution (final concentration 1 mg / ml) of green tea extract at 37 ° C. and Ca ⁺⁺ and collagen were added. 2 ml of TBA reagent was added, warmed at 90 ° C. for 20 minutes, cooled to room temperature, centrifuged at 3,000 rpm for 10 minutes, and the absorbance of the red solution on the settled platelets was measured at 535 nm. The water of the MDA-TBA conjugate was measured. The absorption coefficient was calculated as 1.56 × 10 ⁵ to calculate the amount of MDA produced, and the lipid peroxidation inhibitory activity by green tea extract was compared.

MDA production is used as a measure of total lipid peroxidation as a secondary product of lipid peroxidation. Green tea extract has the same degree compared to aspirin, which inhibits the activity of the phospholipase A2 inhibitors, primaquine, quinacrine and oxygenase. Inhibitory effect on lipid peroxidation was shown at the level of.

b) Determination of arachidonic acid release activity

1 ml of platelet suspension was added to green tea extract (final concentration 1 mg / ml) while standing at 37 ° C. and Ca ⁺⁺ and collagen were added. After 30 minutes, platelets were rapidly removed with a membrane filter, the filtrate was adjusted to acidity of pH 3 or less, extracted twice with 2 ml of extraction solvent (chloroform: methanol = 1: 1 V / V), and centrifuged. After separating, the lower chloroform layer was transferred to another test tube and dried with nitrogen gas. This dried product was dissolved in 50 µl of dimethylformamide (DMF), 5 µl of DMF solution of 12 mM monodansyl cadaverine and 2 µl of diethylphosphoro cyanidate were added, and left at room temperature for 15 minutes to give arachidonic acid. Fluorescent labeling. Fluorescently labeled arachidonic acid was eluted in a reversed phase column by linear gradient elution of 50% methanol: acetonitrile and detected at an excitation wavelength of 340 nm and a fluorescent wavelength of 520 nm.

As a result of examining the quantitative dynamics of arachidonic acid, it was found that the change of arachidonic acid by green tea extract was less than that of primaquine and quinacrine, so that the action site of green tea extracts in the platelet was phospholipase. Inhibition of cyclooxygenase or lipoxygenase, rather than A2, is believed to have an effect of inhibiting lipid peroxidation and platelet aggregation.

6. Treatment effect of hyperlipidemia-antiperoxidation effect (in vitro)

Green tea extract was added to the rat liver microsome fraction (1.5 mg protein / ml), cysteine (final concentration 500 μM) and ferrous sulfate (final concentration 5 μM) were added for 30 minutes at 37 ° C. It was left. 3 ml of 10% trichloroacetic acid was added and centrifuged to give 2 ml of the upper layer, 0.67% TBA solution was added thereto, reacted in boiling water for 15 minutes, and cooled to measure absorbance.

7. Screening-antihypertensive effect (in vitro) on biofunctionality: inhibitory effect of angiotensin converting enzyme (ACE)

Angiotensin converting enzyme (ACE) is an enzyme that acts on the renin-angiotensin system responsible for important regulation of blood pressure, body fluids and electrolytes in vivo. This enzyme releases dipeptides (His-Leu) at the C terminus of angiotensin I (ANG I) produced from renin and converts it to the active angiotensin II (ANG II). The enzyme plays an important role in regulating the circulation of the living body because it produces ANG II of the hypertensive system and inactivates bradykinin of the hypotensive system. Therefore, the search for the inhibitory effect of the green tea extract on this enzyme can determine the degree as a blood pressure increase inhibitor.

Acute Toxicity Experiment

Physiological saline solutions of green tea extracts were administered intravenously and orally, respectively, of ICR mice to calculate LD ₅₀ by life and death determination after 72 hours. The calculation used an up and down method (published in 1969, Namingu, Japan, Kogyo, Kosuke, 204-205). The results are as follows.

As confirmed in the above experiments, the green tea extract of the present invention was found to be extremely low acute toxicity.

The green tea extract of the present invention may be dosed once or several times daily between 1.00-2000.00 mg per kg of body weight, depending on the age, sex, symptoms or prevention of the patient or person to be administered.

The green tea extract of the present invention may be mixed with conventional auxiliaries such as excipients, binders, lubricants, disintegrants, diluents and the like to prepare pharmaceutical preparations.

The following formulation examples are illustrated.

Preparation Example 1 Preparation of Tablet

Green Tea Extract 10mg

Lactose 20mg

Starch 20mg

Magnesium stearate equivalent

Tablets were prepared by tableting the above components with 50 mg of tablets according to a conventional tablet production method.

Preparation Example 2 Preparation of Capsule

Green Tea Extract 10mg

Lactose 20mg

Starch 19mg

Talc 1mg

Magnesium stearate equivalent

The above ingredients were usually filled in a 50 mg capsule according to the method for preparing a capsule.

Preparation Example 3 Preparation of Liquid

Green Tea Extract 100mg

10 g of isomerized sugar

Honey 500mg

Niacinamide (Pharmacopoeia) 20mg

Caffeine anhydrous 30mg

Sodium Benzoate 70mg

The above ingredients were prepared according to a conventional liquid preparation method, and filled into 100 ml brown bottles, tightly pasteurized and pasteurized.

Preparation Example 4 Preparation of Injection

Green Tea Extract 5mg

Sterile water purification

The above ingredients were prepared according to a conventional injection method, and filled in 2 ml ampoules and sterilized by sealing.

Preparation Example 5 Preparation of Soft Capsule

1) Filling composition

Green Tea Extract 10mg

Polyethylene Glycol 230mg

Glycerin 13mg

2) Dry skin composition (% by weight)

52% gelatin

Glycerin 32%

ANIDRISORB 35/7012%

5% of water

The above compositions were prepared in a soft capsule according to a conventional soft capsule manufacturing method.

Preparation Example 6 Preparation of Granules

Green Tea Extract 10mg

Lactose 25mg

The above components were extruded with a conventional extrusion granulator to prepare granules.

The antithrombotic green tea extract of the present invention may be used as a composition mixed with a pharmacological substance having another conventional medicinal effect. Such compositions are, of course, also within the scope of the present invention.

[Effects of the Invention]

As described and demonstrated in detail above, the green tea extract of the present invention comprising 25-30% of polyphenols, 40-70% of epigallocatechin gallate and 0-35% of a mixture of epicatechin and epigallocatechin promotes various aggregations. Not only does it exhibit better platelet aggregation inhibitory effects than aspirin, which has side effects of gastrointestinal disorders, but also anti-thrombotic and anticoagulant properties in vivo, and therefore, it is highly desirable as an antithrombotic agent. In addition, by extracting the chloroform extraction process in the preparation of the electrical extract, by employing column chromatography using a mixed solution of water and acetone, dioxane, methanol or isopropanol as a specific separation solvent, a green tea extract having a composition unique to the present invention is prepared. .

Claims (2)

The dried green tea leaves were extracted with hot water with water or a mixture of water and lower alcohol, filtered or centrifuged to remove non-polar substances, and the organic solvent layer obtained by repeated extraction with ethyl acetate was extracted with water, acetone, dioxane, methanol or isopropanol. 25-30% of polyphenols, 40-70% of epigallocatechin gallate, and epicatechin and epigallocatechin, characterized in that the active ingredient obtained by column chromatography was lyophilized using a mixed solution of Method for producing a green tea extract consisting of 0 to 35% of the mixture. In the green tea extract contained as an active ingredient in the antithrombicide, the composition of the electric green tea extract is 25 to 30% of polyphenols, 40 to 70% of epigallocatechin gallate and 0 to 35% of a mixture of epicatechin and epigallocatechin. Green tea extract, characterized in that consisting of.